The ability of cells to utilize cholesterol derived from lipoprotein is important in normal steroidogenesis, membrane biosynthesis, and regulation of sterol synthesis. Pathological accumulation of esterified cholesterol is a central feature of atherosclerosis. While the endocytosis of lipoprotein derived cholesterol has been well characterized, the subsequent events that mediate the post-lysosomal intracellular transport of cholesterol derived from lipoprotein are not understood. This proposal presents evidence that the capacity of human adrenal tumor cells to esterify lipoprotein derived cholesterol is affected by vimentin-type intermediate filaments (IFs), a major component of the cytoskeleton. Preliminary studies indicate that the intracellular transport of lipoprotein-derived cholesterol from the lysosome to the site of esterification in these cells is a vimentin IF dependent process, but the mechanism is unknown. In the proposed experiments, human SW-13 adrenal tumor cell lines that either contain vimentin-type IFs, or completely lack any detectable IF network will be studied to define and characterize the step in the intracellular transport of cholesterol that is affected by vimentin IF expression. The specificity of vimentin effects on cholesterol transport in these studies will be established by examination of stable cell lines derived from IF negative cells that express a mouse vimentin cDNA, and cell lines with IF networks specifically disrupted by expression of a dominant negative mutant vimentin. Studies following the fate of radiolabeled LDL-cholesteryl linoleate will determine whether IFs affect cholesterol movement from the lysosome to the plasma membrane, from the plasma membrane to the site of esterification, or perhaps from the lysosome to the site of esterification by a mechanism that can bypass the plasma membrane. The intracellular movement of a fluorescent cholesteryl ester will also be studied in cells that contain or lack IFs to follow the intracellular movement of cholesterol from the lysosome using fluorescence microscopy. Adrenal tumor cell lines that express keratin-type IFs will be derived from cells that do not express endogenous cytoplasmic IFs by stable transfection with keratin cDNA expression plasmids. The ability of these cells to esterify lipoprotein derived cholesterol will be compared with cell lines that contain or lack vimentin IFs to determine whether keratin IFs, characteristic of epithelial cells, have a similar function. The expression and cytoplasmic localization of sterol carrier protein-2 (SCP- 2), and apolipoprotein E (apoE) will also be examined in SW-13 cells that contain or lack IFs. Studies will be conducted using immunoprecipitation and immunofluorescence microscopy with anti-SCP-2 and anti-apoE antibodies to determine if the effect of IFs on the transport of lysosomal cholesterol could involve an association with either SCP-2 or apoE. The long-term goal of this project is to define and understand the molecular details of this process, its regulation, and potential involvement in steroidogenesis and pathological cholesterol accumulation.